ABSTRACT Despite unprecedented gains in the basic understanding of cancer genes, only a few molecular therapies have become standard of care in thirty years. This may reflect the extraordinary molecular heterogeneity of human tumors, but also a relatively narrow path to cancer drug discovery, which typically focuses on individual drugable targets in isolation. Conversely, agents capable of disabling multiple essential networks of tumor maintenance, i.e. pathway inhibitors, may offer broader therapeutic opportunities. One attractive candidate for this approach is the molecular chaperone Heat Shock Protein-90 (Hsp90), which, together with its related molecules, orchestrates pivotal cancer networks of cell proliferation, survival and adaptation. Recently, we identified Shepherdin, a novel peptidomimetic inhibitor of Hsp90. Compared to other Hsp90 antagonists currently in the clinic, Shepherdin exhibits superior anticancer activity in vitro and in vivo, efficacy against heterogeneous tumor cell types regardless of their genetic makeup, and no toxicity for normal tissues. New experimental evidence has now uncovered a complex Shepherdin pathway, underlying this unique anticancer activity. We found that Shepherdin acts on mitochondria, inducing sudden organelle collapse and cell death. At a molecular level, this is due to the inhibition of a pool of Hsp90 chaperones present in mitochondria of tumor cells, and orchestrating a novel pathway of cell survival. Therefore, the hypothesis that Shepherdin is a novel pathway inhibitor with a unique mechanism of anticancer activity can be formulated, and will constitute the focus of the present resubmitted application. Experiments in the first specific aim will characterize the dynamics of Shepherdin trafficking and import to tumor mitochondria, and dissect the molecular requirements for activation of organelle dysfunction. The second specific aim will elucidate the survival functions of a mitochondrial Hsp90 network in tumor cells, and map their interactions with the molecular machinery of organelle homeostasis. The third specific aim will test the efficacy of Shepherdin in xenograft and transgenic models of drug-resistant tumors, metastasis, angiogenesis, and eradication of cancer stem cells, in vivo. The approach combines mechanistic and late-preclinical studies designed to elucidate the Shepherdin pathway and its anticancer activity. The results will complement the clinical development of Shepherdin now underway at the National Cancer Institute (NCI), Rapid Access to Intervention Development (RAID) program, and will provide a requisite molecular foundation to direct the upcoming testing of this agent in humans.